Osteoporosis is a major public health problem, especially in women. It is mainly characterized by low bone mineral density (BMD). BMD has high heritability of >60%. Women have much lower BMD than men. We and others demonstrated that some BMD genes/genomic regions are gender specific. Menopause is a most significant physiology event in female's life and is associated with female-specific rapid bone loss. Bone marrow mesenchymal stem cells (BMMSCs) and peripheral blood monocytes (PBMs), are precursors for osteoblasts (bone formation cells) and osteoclasts (bone resorption cells), respectively. Our hypothesis is that changes in the protein expression profiles in female BMMSCs and PBMs underlie mechanisms of female BMD variation and are associated with menopause. Our major goals here are to identify proteins differentially expressed in BMMSCs and PBMs in women: 1) with high vs. low BMD;2) before and after menopause, and thus identify proteins (and their genes) associated with female osteoporosis and menopause in BMMSCs and PBMs. The proteins identified significant in females will be examined in male samples. Together with Project 2, we will recruit 80 otherwise healthy females and 80 age-matched otherwise healthy males aged 50-55, including 40 subjects with low and 40 with high BMD (age matched population bottom or top 20% respectively) for each sex, all Caucasian. Each female BMD group includes 20 pre- and 20 age matched post-menopausal women. We will take fresh bone marrow (for which we have had extensive published research experience) and peripheral blood samples from each subject. BMMSCs and PBMs will be isolated and equally divided into two aliquots, one for Project 2 and one for Project 3. In this Project 3, we will extract the total proteins from aliquots of isolated BMMSCs and PBMs. Proteomic profiling experiments and analyses will be performed on Females using MD-nano-LC-MS/MS. Significant differentially expressed proteins identified will be verified by Western blotting with female samples for their importance in females. Significant proteins verified in females will be examined by Western blotting with male samples for sex-specificity. The results obtained in Caucasians will be cross checked for ethnic generality/specificity in Chinese samples. The molecular and cellular functional studies of the identified proteins and their genes will be pursued as exemplified in Project 2 of this SCOR. The major results (particularly those obtained from PBMs) of this study may be used to design customary diagnostic protein antibody chips and/or protein markers for prognosis of female osteoporosis. In particular, the results will be used to provide some functional evidence, when combined with the DMA polymorphism data in Project 1 and DMA microarray data in Project 2 of this SCOR to powerfully and efficiently identify genes and some of their functions for female osteoporosis.